POS0441 IN-DEPTH ANALYSIS OF Axl AND MerTK EXPRESSION PATTERNS AND REGULATION BY BIOLOGIC TREATMENTS IN RHEUMATOID ARTHRITIS
Authors: A. Nerviani, M. A. Boutet, G. M. Ghirardi, K. Goldmann, E. Sciacca, F. Rivellese, E. Pontarini, M. Caliste, E. Prediletto, M. Bombardieri, M. Lewis, C. Pitzalis
Journal: Annals of the Rheumatic Diseases (2022)

Background Tyrosine kinases receptors MerTK and Axl have been implicated in the pathogenesis of several autoimmune diseases. Despite sharing significant structural homology and having common ligands, Axl and MerTK have distinct features and biological functions [1]. A growing body of evidence suggests that both Axl and MerTK play a crucial role in Rheumatoid Arthritis (RA) pathogenesis and progression and may be exploited as novel therapeutic targets [2]. However, numerous unanswered questions remain to be addressed. Objectives: i.To define common and distinct gene-partners of Axl/MerTK and quantify their expression in RA synovial tissue. ii.To assess the co-expression of Axl/MerTK by synovial cells. iii.To outline the longitudinal variation in Axl/MerTK expression upon treatment intervention. Methods Synovial tissue samples were collected by US-guided synovial biopsy from: i. Patients with early (&lt;12 months) RA DMARDs/steroid-naïve [n=87]; and ii. RA patients who failed the first-line biologic with TNF-inhibitors (TNFi) before and 16 weeks after receiving either Rituximab (RTX) or Tocilizumab (TOC) [n=164] [3]. Gene expression was obtained by bulk RNAseq performed on an Illumina HiSeq2500 platform. Axl-/MerTK-modules were defined using STRING networks and the module expression determined by the mean z-score of regularized log transformed expression for all genes in the set. Axl, MerTK, CD55, CD90, CD68 protein expression was analysed by multiplex immunofluorescence staining. Results Using STRING network analysis, we defined an Axl- and a MerTK-module composed of 31 predicted gene-partners of either Axl or MerTK. Thirteen genes were common to both modules and included the ligands Gas6 and ProteinS, and EGFR. Conversely, eighteen genes were uniquely present in the Axl-module (e.g., PIK3-family, IGF1R, IFNAR1 and STAT3) or the MerTK-module (e.g., Galectin3 and TULP, recently discovered MerTK ligands, FCGR1A/CD64, PTPN1and MEGF10). Axl/MerTK-modules quantified in the early-arthritis treatment-naïve RNAseq dataset showed a significant negative correlation with the synovitis score (Axl r=−0.33, p=0.0032; MerTK r=-0.33, p=0.003). At protein level, CD68+macrophages of the Lining showed notable heterogeneity between patients: they could express either Axl or MerTK alone, or co-express both. Axl was also present in most CD55+ Lining Fibroblast-Like-Cells (FLS) but not by CD90+ Sublining FLS while MerTK, as expected, was restricted to macrophages, including intra-aggregate tingible-body-macrophages. To define how Axl and MerTK vary depending on disease stage and treatment exposure, we quantified their gene expression in active RA patients inadequately responding to TNFi, prior and 16 weeks after starting second-line biologic (RTX or TOC) [3]. Differently from the early-arthritis cohort, MerTK was significantly up-regulated in synovia characterised by higher degree of tissue inflammation (lympho-myeloid &gt; diffuse-myeloid &gt; pauci-immune, p&lt;0.0001) and significantly positively correlated with several cytokines’ genes such as TNF, IL-6, CCL8 and IL-10. MerTK expression was dependent on clinical response to RTX but not TOC as assessed by EULAR response (DAS28CRP, good vs none/mod, FDRresp 0.048). Conversely, Axl expression significantly increased upon IL-6 blockade by TOC independently of the clinical response (FDRtime 0.016). Conclusion Our data further corroborate that Axl and MerTK constitute a dynamic axis influenced by the synovial tissue inflammatory features, the disease stage, the exposure and the response to targeted treatment and the blockade of critical inflammatory pathways over time. A better understanding of the individual features of these tyrosine kinases as well as their interaction would be beneficial to define novel treatment approaches. References [1]Zagórska A, et al. Nat Immunol. 2014 Oct;15(10):920-8 [2]Kemble S, Croft AP. Front Immunol. 2021 Sep 3;12:715894 [3]Humby F et al. Lancet. 2021 Jan 23;397(10271):305-317 Acknowledgements Versus Arthritis. Disclosure of Interests None declared.